Design, Synthesis, and Biological Evaluation Studies of Novel Naphthalene-Chalcone Hybrids As Antimicrobial, Anticandidal, Anticancer, and VEGFR-2 Inhibitors

Cancer is a progressive disease that is frequently encountered worldwide. The incidence of cancer is increasing with the changing living conditions around the world. The side-effect profile of existing drugs and the resistance developing in long-term use increase the need for novel drugs. In addition, cancer patients are not resistant to bacterial and fungal infections due to the suppression of the immune system during the treatment. Rather than adding a new antibacterial or antifungal drug to the current treatment plan, the fact that the drug with anticancer activity has these effects (antibacterial and antifungal) will increase the patient’s quality of life. For this purpose, in this study, a series of 10 new naphthalene-chalcone derivatives were synthesized and their anticancer-antibacterial-antifungal properties were investigated. Among the compounds, compound 2j showed activity against the A549 cell line with an IC50 = 7.835 ± 0.598 μM. This compound also has antibacterial and antifungal activity. The apoptotic potential of the compound was measured by flow cytometry and showed apoptotic activity of 14.230%. The compound also showed 58.870% mitochondrial membrane potential. Compound 2j inhibited VEGFR-2 enzyme with IC50 = 0.098 ± 0.005 μM. Molecular docking studies of the compounds were carried out by in silico methods against VEGFR-2 and caspase-3 enzymes.


INTRODUCTION
The disorder known as cancer, which can arise from any cell and develop anywhere in the body, is spreading across the world. Cancer is the most serious condition, according to statistics and the growth of cancer cells. 1 With an estimated 1.8 million deaths (18%), lung cancer remained the most common type of cancer death in 2020, followed by colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast (6.9%) cancer. Globally, it was estimated that approximately 19.3 million new cancer cases and nearly 10 million cancer deaths occurred in 2020. 2 The potential of developing cancer is increased by a number of risk factors, including alcohol and cigarette use, exposure to carcinogens, obesity, and family history. 3 Cancer presents a significant challenge to the medical research community in terms of developing treatments, therapies, and strategies for more efficient care for patients. 4 Naphthalene ring is a ring rich in biological activity. There are many studies in the literature on the anticancer activity of naphthalene ring. 5 There are also many studies on the antimicrobial activity of this ring. 6 Although chalcones are α, β unsaturated ketone compounds in a 1,3-diaryl-2-propenone structure, their importance in medicinal chemistry is increasing due to their easy synthesis and simple chemistry. The synthesis of chalcones is carried out by different reaction types, such as Claisen−Schmidt reactions, Heck coupling, Suzuki−Miyaura coupling, and Witting reactions. They are compounds with very wide biological activity according to their substitutions in the aryl structure. Examples of these are anti-inflammatory, antimicrobial, anticancer, cytotoxic, and anticholinesterase. 7 In addition, there are many publications showing the anticancer or antimicrobial activities of chalcones. 8 In cellular activities, such as growth, survival, invasion, and angiogenesis during tumor initiation and development, protein kinases play different signaling pathways. 9 The initial tumor and any resulting metastases are dependent on angiogenesis, according to recent research. Several protein kinases, including the growth factors, regulate the angiogenesis process. The vascular endothelial growth factor (VEGF) is one of the most potent angiogenic determinants that can control angiogenesis and be involved in the development of a tumor among those growth factors. 10 By interacting with VEGF receptor types 1− 3, the VEGF family controls angiogenesis. 11 The VEGFR-2 binding site is hydrophobic, hence VEGFR-2 inhibitors showed a wide variety of chemical configurations. However, there were significant similarities between the chemical structures of the two well-known inhibitors (sorafenib I and regorafenib II) that are necessary for any inhibitor to correctly engage with the active binding site. 12 In light of the above information, 10 new compounds were synthesized within the scope of this study. The naphthalene ring of the compounds was used as the bioisostere of the quinoxaline ring in the structures of the third generation VEGFR inhibitor Lenvatinib and Cabozantinib compounds ( Figure 1). The piperazine ring was preferred because of the secondary amine derivatives in Sunitinib and Nintedanib, which are second generation VEGFR inhibitors.

RESULTS AND DISCUSSION
2.1. Chemistry. The compounds 2a−2j were synthesized as presented in Scheme 1. First, the ketone derivatives (1a− 1e) were obtained by using 4-fluoroacetophenone and appropriate secondary amine. Between 1-naphtaldehyde or 2naphtaldehyde and the ketone derivatives (1a−1e), Claisen Schmitt reaction was carried out for obtain target compounds (2a−2j). Spectroscopic techniques, such as 1 H NMR, 13 Table  1. None of the compounds showed activity against the HepG2 cell line. Among the compounds, only compound 2j has an IC 50 below 10 μM. Compound 2j showed activity against A549 cell line with the value of IC 50 = 7.8 ± 0.59 μM. In addition, compounds 2e and 2i showed activity against the A549 cell line with IC 50 = 20.6 ± 0.52 μM and IC 50 = 21.4 ± 2.6 μM, respectively. It is important that a compound does not show cytotoxic activity on a healthy cell, as well as showing activity on cancer cells. For this purpose, the cytotoxic effects of the compounds against the healthy mouse fibroblast cell (NIH3T3) were investigated. The most active compound, compound 2j, showed cytotoxicity against the NIH3T3 cell line with an IC 50 = 15.6 ± 0.8 μM. Calculating the selectivity index for this compound (IC 50 value vs healthy cell/IC 50 value vs cancer cell) is 1.990 against A549 cell line. Further testing was performed for compound 2j, the most active compound in the series. The apoptotic potentials and anticancer activity mechanism of this compound were tried to be clarified.
When the structures of the compounds are examined, it is seen that there are common naphthalene and piperazine rings in all compounds. The naphthalene ring is substituted from the first position (2a−2e) in some compounds and from the second position in some compounds. In the fourth position of the piperazine ring, compounds 2a and 2f contain methyl; compounds 2b and 2g contain ethyl; compounds 2c and 2h contain isopropyl; compounds 2d and 2i contain allyl; and compounds 2e and 2j contain 2-methoxyethyl substituent.
When the structure−activity relationships are examined, it is seen that the substitution of the naphthalene ring from the second position increases the activity. In addition, the idea that naphthalene ring contributes positively to the activity is strengthened by the aromatic hydrogen bonds it forms in the enzyme active sites. The incorporation of the 2-methoxyethyl substituent into the structure increases the activity. The two most active compounds in the series are the compounds that carry the 2-methoxyethyl substituent (2e and 2j). MIC 50 values were determined via fluorometric measurements, using resazurin solution. 13 Azithromycin was used as a standard drug in the antibacterial activity test. Results are presented in Table 1. When the antibacterial activity profile was examined, compound 2f showed the highest activity against S. epidermis with a value of MIC 50 = 15.6 μg/mL. Moreover, compounds 2d and 2j displayed the highest activity against E. faecalis with a value of MIC 50 = 15.6 μg/mL.
Obtained compounds (2a−2j) were evaluated for anticandidal activity against Candida albicans (C. albicans) (ATCC 24433), Candida krusei (C. krusei) (ATCC 6258), and Candida parapsilopsis (C. parapsilopsis) (ATCC 22019). MIC 50 values were determined via fluorometric measurements, using resazurin solution. 13 Voriconazole and fluconazole were used as a standard drug in the anticandidal activity test. Results are presented in Table 1. When the anticandidal activity profile was examined, compounds 2b, 2c, 2e, and 2j showed the highest activity against C. albicans with a value of MIC 50 = 15.6 μg/mL. Additionally, compound 2j displayed the highest activity against C. krusei with a value of MIC 50 = 15.6 μg/mL. In general, it is seen that the 2-methoxyethyl substituent increases the anticandidal activity. When the substitution of naphthalene in its first position and its substitution from its second position is compared, the derivative containing 2substituted naphthalene (compound 2j) showed activity on two Candida lines.
2.4. Flow Cytometric Analysis. Using a flow cytometer, gating was performed on a particular labeled cell, designated as Compound 2j caused 18.1% necrosis, 10.3% late apoptosis, and 3.97% early apoptosis. All cells exposed to the chosen substance have been seen to activate the apoptotic process. Taking into account all flow cytometry findings, it was found that 2j was the molecule that caused both cell types to undergo the maximum apoptosis. Compared to the SD4, they triggered apoptosis at a similar rate (Doxorubicin). The rate of apoptosis caused by compound 2j was 14.2% (3.97% early apoptosis, 10.3% late apoptosis), while the rate induced by SD4 was 17.4% (8.55% early apoptosis, 8.84% late apoptosis). However, compound 2j also increased the likelihood of necrosis by 18.1%.

Analysis of Mitochondrial Membrane Potential (MMP) by Flow Cytometry.
Mitochondria-targeted agents play a very important role in the eradication of chemotherapyresistant cancer cells. The most important reason for this is that mitochondria are key regulators of cell death. In addition, frequent changes in mitochondrial functions in neoplasia bring mitochondria-targeted drugs to the fore. 14 For this purpose, the mitochondrial membrane potential of compound 2j (the most active compound) was determined against A549 cell line by in vitro flow cytometric methods. Compound 2j and doxorubicin were applied at IC 50 concentration. After a 24-h incubation period, mitochondrial membrane potential was measured with JC-1 dye. The results obtained with the control group, compound 2j and doxorubicin are presented in Figure  3. According to the results obtained, while doxorubicin showed 30.270% mitochondrial membrane potential; compound 2j showed 58.870% mitochondrial membrane potential.
2.6. VEGFR-2 Inhibition Assay. The VEGFR-2 Kinase Assay Kit (Available from ref 15) was used for the VEGFR-2 inhibition. The experiment was performed in vitro according to the kit procedure. Serial 11 dilutions of compound 2j were prepared at concentrations of 1000 μM−0.01 μM. The IC 50 value for compound 2j was calculated using the kit procedure. According to the results obtained, the compound 2j shows inhibitory activity on VEGFR-2 enzyme with the value of IC 50 = 0.098 ± 0.005 μM.

In Silico Study.
To justify potency improvement of the novel synthesized naphthalene-chalcone derivatives (2a−2j), molecular docking was conducted to investigate the potential binding mode of the most potent inhibitor (compound 2j). Docking studies were performed on the VEGFR-2 crystals (PDB ID: 4ASE and PDB ID: 4ASD), 16 as well as Caspase-3 (PDB ID: 4QTX), 17 to demonstrate the caspase contribution of the apoptotic pathway.
Compound 2j's location in the VEGFR-2 enzyme's active region is shown in Figure 4A. (PDB ID: 4ASE). The interaction with amino acids in the active site is shown in Figure 4B. Examining these interactions reveals a hydrogen connection between the amino group of Cys919 and the carbonyl group of molecule 2j. The hydroxyl group of Asp1046 creates a salt bridge with the terminal nitrogen of the piperazine ring. The amine group of Glu885 and the unpaired electrons of the oxygen atom of the 2-methoxyethyl substituent form a hydrogen bond. In addition, the carbonyl groups of Leu840 and Lys920 were joined by aromatic hydrogen bonds between the hydrogens of the naphthalene ring.
The location of compound 2j in the VEGFR-2 enzyme's active region is depicted in Figure 4C. (PDB ID: 4ASD). The interaction with amino acids is depicted in Figure 4D for the active site. The naphthalene in compound 2j establishes two   The test results were expressed as means of triplicate assays. b The test results were expressed as means of triplicate assays. c The test results were expressed as means of quartet assays ± SEM. SD1: Azithromycin. SD2: Voriconazole. SD3: Fluconazole. SD4: Doxorubicin.
pi-pi connections with the amino group of Lys838, according to the analysis of these interactions. The oxygen atom of the 2methoxyethyl substituent's unpaired electrons and the Asp1046 amine combine to create a hydrogen bond. Additionally, aromatic hydrogen bonds were created between the carbonyl groups of Lys920 and Cys919 by the hydrogens on their respective naphthalene and phenyl rings. Compound 2j's location within the Caspase-3 enzyme's active region is seen in Figure 4E. The interaction with amino acids in the active site is depicted in Figure 4F. When these interactions are analyzed, it becomes clear that the amino group of Arg207 and the oxygen atom of the 2-methoxyethyl substituent create a hydrogen bond. With the amino group of Arg207, the 1,4-disubstituted benzene interacts via a cation-pi interaction. With Trp206's indole ring, the same ring interacts in a pi−pi fashion. Naphthalene ring hydrogens also created an aromatic hydrogen connection with Glu248's carbonyl group.

CONCLUSION
The morbidity and mortality rates of cancer disease are increasing day by day. The side-effect profiles of the drugs used in the treatment and the resistance to the drugs create a need for new and effective drugs. Tyrosine kinase inhibitors have become a major part of cancer treatment in recent years. The advantages of the patient such as ease of use and targetoriented treatment have brought this group of drugs to the fore. For this purpose, within the scope of this study, new naphthalene-chalcone derivatives were synthesized and their anticancer effects were investigated. During cancer treatment,  patients become vulnerable to bacterial and fungal infections. For this purpose, the fact that a drug with anticancer activity also shows antibacterial and anticandidal activity makes that drug more valuable.

General Procedures of Target Compounds (2a−2j).
The %40 KOH solution was prepared in MeOH (>99.5%). Appropriate obtained ketones (0.001 mol) were dissolved in the solution of KOH, and the obtained reaction mixture was stirred in room temperature for 30 min. Then the aldehydes (1-naftaldehyde or 2-naftaldehyde) were added in reaction mixture. After completion of the reaction, the precipitated product was filtered, washed with MeOH, and dried.    126.2, 126.8, 128.1, 128.7, 130.4, 130.8, 131.8, 132.9 113.3, 113.6, 122.1, 123.8, 126.7, 127.2, 127.8, 128.3, 128.6,  130.3, 130.7, 132.8, 133.4, 134.2, 143.3, 154.1, 188     The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt reduction method, or MTT assay, is used to measure the metabolic activity of live cells. The formazan salt, which turns purple at the conclusion of the incubation period, allows for the spectrometric determination of the cell viability rate. 18 Table 1 shows findings of cell growth inhibition following a 24-h treatment with the resulting compounds for 4 different cell lines. MTT assays were used to screen compounds 2a−2j for cytotoxicity. Doxorubicin is used as a therapeutic drug. MTT assays were carried out as previously explained. 19 4.3. Antibacterial and Anticandidal Activity. The antimicrobial activity of obtained derivatives (2a−2j) was screened on six bacterial and three fungal strains according to the standard procedure of CLSI 20 as described in the previous study. 21 The antibacterial activities of the obtained compounds were screened against B. subtilis (ATCC 6051), E. coli (ATCC 25922), E. faecalis (ATCC 2942), P. aeruginosa (ATCC 27853), S. aureus (ATCC 29213), and S. epidermidis (ATCC 12228). The obtained compounds were evaluated for anticandidal activity against C. albicans (ATCC 24433), C. krusei (ATCC 6258), and C. parapsilopsis (ATCC 22019). For antibacterial activity, azithromycin was utilized as a reference drug, whereas voriconazole and fluconazole were used for anticandidal activity. 4.6. In Silico Study. Molecular docking studies were performed using in-silico procedure to define the binding modes of compound 2j (active compound) in the active regions of enzymes X-ray crystal structures of VEGFR-2 (PDB ID: 4ASE and PDB ID: 4ASD) 16 and Caspase-3 (PDB ID: 4QTX) 17 were retrieved from Protein Data Bank server (www. pdb.org, accessed 19 Sep 2022). The Schrodinger Maestro interface, 24 Ligprep module, 25 and Glide module 26 were used for molecular docking procedures, and docking runs were performed in standard precision docking mode (SP).

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c07256. 1 H NMR spectra of the compound 2a; 13 C NMR spectra of the compound 2a; HRMS spectra of the compound 2a; 1 H NMR spectra of the compound 2b; 13 C NMR spectra of the compound 2b; HRMS spectra of the compound 2b; 1 H NMR spectra of the compound 2c; 13 C NMR spectra of the compound 2c; HRMS spectra of the compound 2c; 1 H NMR spectra of the compound 2d; 13 C NMR spectra of the compound 2d; HRMS spectra of the compound 2d; 1 H NMR spectra of the compound 2e; 13 C NMR spectra of the compound 2e; HRMS spectra of the compound 2e; 1 H NMR spectra of the compound 2f; 13 C NMR spectra of the compound 2f; HRMS spectra of the compound 2f; 1 H NMR spectra of the compound 2g; 13 C NMR spectra of the compound 2g; HRMS spectra of the compound 2g; 1 H NMR spectra of the compound 2h; 13 C NMR spectra of the compound 2h; HRMS spectra of the compound 2h; 1 H NMR spectra of the compound 2i; 13 C NMR spectra of the compound 2i; HRMS spectra of the compound 2i; 1 H NMR spectra of the compound 2j; 13 C NMR spectra of the compound 2j; and HRMS spectra of the compound 2j (PDF)